ABSTRACT Polymicrobial infections are difficult to treat because of either inherent or acquired resistance of microbes to different classes of antibiotics. During polymicrobial infections, synergistic augmentation of the virulence potential of pathogens is a significant concern with devastating patient outcomes. While mixed infections may represent association of diverse groups of bacteria, mutualistic, synergistic, or antagonistic relationships can occur between bacteria, fungi, and viruses. Alarmingly, how multispecies interactions govern progression and disease severity and how the host responds to such polymicrobial or monomicrobial infections are poorly understood. While studying a human case of necrotizing fasciitis (NF), we identified Aeromonas hydrophila to be the causative agent and the infection was considered monomicrobial. However, after deep sequencing of DNA from infected tissues and bioinformatics analysis, we determined that the infection, in fact, involved four strains of the same pathogen with differing virulence; three strains (designated as NF2, NF3, and NF4) were clonal, while the fourth strain, NF1, was phylogenetically distinct. Thus, a classically defined monomicrobial infection (a single species) may actually be polymicrobial when different strains of the same bacterial species are present. This concept extends the classical Koch?s postulates to now encompass monomicrobial and polymicrobial infections caused by either (i) a pure culture or (ii) a mixture of bacterial strains of the same species. To our knowledge, we have identified for the first time a toxin (ExoA) that is responsible for synergistic/crosstalk events in mono-species polymicrobial strain infection. In addition, the presence of a type 6 secretion system (T6SS) effector and immunity protein was noted in NF1 based on genome analysis which was absent from the strain NF2. Our results of mixed infection in a mouse model of NF clearly indicated interactive processes of both synergistic augmentation and antagonistic attenuation of virulence of two distinct strains, NF1 and NF2. However, the molecular mechanisms governing such cross-talk among strains with differing genome signatures are poorly understood and will be explored. In Aim 1, the role of the type 6 secretion system and its effector TseC and immunity protein TsiC in contact-dependent killing of NF2 by NF1, the latter of which only harbors this effector:immunity protein pair, will be evaluated with both in vitro and in vivo models using metagenomics tools and luciferase-marked strains. In Aim 2, the role of ExoA (of NF2) in modulating bacterial motility to facilitate better dissemination of NF1 (without ExoA) in an in vivo model of NF will be delineated. Further, protective effects of ExoA antibodies in mice against development of NF will be investigated. The overall significance of our study also translates to many other diseases which are polymicrobial in nature. Importantly, development of alternate therapeutics to combat bacteria with distinct antibiotic profiles in the context of mono-species polymicrobial infections is critical to prevent devastating disease sequelae. The central question of how one strain promotes the spread of the second strain, and how the second strain promotes clearance of the first is very exciting and forms the basis of our study.